The MAGT1 transporter is critically involved in the selective regulation of intracellular free Mg2+ levels in mammalian cells. The molecular functions of free Mg2+ in eukaryotic cells have not been fully established. We found that patients with genetic deficiencies in MAGT1 have high levels of Epstein-Barr virus (EBV) and a predisposition to lymphoma. In studying lymphocytes from these patients, we found that a deficiency of MAGT1 caused decreased basal intracellular free Mg2+ leading to defective expression of the natural killer activating receptor NKG2D in NK and CD8+ T cells. Without NKG2D, cytolytic responses against EBV are diminished, thereby revealing the first specific molecular function of intracellular basal free Mg2+ in eukaryotic cells. Moreover, intracellular free Mg2+, NKG2D expression and function can be rescued in vitro by incubating patient cells and elevated levels of Mg2+. Moreover, NKG2D expression and cytolytic function can be improved and EBV-infected cells reduced in vivo, in MAGT1-deficient patients by magnesium administration. Thus, our data indicate an important molecular function for free basal Mg2+ in immunity and demonstrate a requirement for NKG2D cytolytic function in an essential EBV antiviral response in humans. We are especially interested in pursuing additional questions related to the role of Mg2+ in the control of EBV. Despite being linked to both epithelial (nasopharyngeal and gastric) and lymphoid (Burkitt and Hodgkin lymphoma) malignancies, there are currently no known methods for primary or secondary prevention of chronic EBV infection or the associated malignancies. Our discovery that a genetic deficiency of a Mg2+ ion transporter caused a selective immunodeficiency that led to uncontrolled EBV infection and an extremely high rate of EBV+ lymphoma in affected children and that dietary supplementation with Mg2+ (a widely available and inexpensive nutraceutical) could correct the immune defect by increasing a specific antiviral receptor called NKG2D which markedly decreased or eliminated EBV offered a new hypothesis about chronic EBV in Africa. We have begun a collaboration with Sam Mbulaiteye, a National Cancer Institute investigator, who studies EBV/lymphoma risk in Africa. We have carried out a preliminary study of previously collected case-control blood samples from Africa and showed that there was a statistically significant deficiency of serum Mg2+ in Burkitt lymphoma (BL) patients with high EBV. This preliminary study could not answer whether intracellular Mg2+ and NKG2D expression were deficient in these patients which requires flow cytometric analysis on site in Africa or whether these could be restored by adding more Mg2+ to the cells. However, these results held promise that endemic EBV and the consequent lymphomas could be prevented by simple dietary supplementation with Mg2+. In an effort to understand the molecular mechanism by which alterations in free Mg2+ affect immune signaling, we carried out a detailed study in human T lymphocytes. We found that extracellular Mg2+ can profoundly affect activation and proliferation of T cells due to the fact that intracellular free Mg2+ is in equilibrium with extracellular Mg2+. We are able to accurately measure this using a null point titration assay adapted for precise Mg2+ measurements. Chronic Mg2+ deprivation in T cell blasts reduced intracellular Mg2+ levels and impaired Ca2+ fluxes upon T cell receptor (TCR) stimulation. Acute Mg2+ withdrawal at the time of TCR stimulation also caused Ca2+ flux defects and reduced phosphorylation of signaling proteins downstream of inducible T cell kinase (ITK). We show that ITK activity is enhanced by Mg2+ at physiologically relevant concentrations and molecular modeling of the ITK structure is consistent with the presence of a second regulatory Mg2+ in the catalytic pocket of ITK. Additional analyses support the idea that this is a conserved feature of many kinases. Finally, perturbation of Mg2+ homeostasis in mice results in an impaired CD8+ T cell response to influenza A virus. In conclusion, our evidence shows that Mg2+ is a regulator of T cell responses and dietary Mg2+ intake can directly affect immunity in healthy individuals.